The Notch signaling pathway and its components are highly conserved in all Metazoans, from Drosophila to humans. The signaling pathway comprises the Notch receptors and their ligands, all single-pass transmembrane proteins with conserved protein structures (Lardelli, 1994, Mech. Dev. 46, 123-1365). Additional elements of the pathway include positive and negative modifiers as well as transcription factors. Notch receptors transduce essential developmental signals between neighboring cells by forming a complex that leads to the transcription of target genes upon activation. Neighboring cells expressing various Notch ligands and receptors amplify and consolidate molecular differences, eventually dictating cell fates and implementing developmental programs of differentiation, proliferation and apoptosis.
In mammals, Notch genes encode four large Type I transmembrane proteins called Notch receptors (Notch 1, Notch 2, Notch 3 and Notch 4) comprised of multiple known structural motifs. En route to the cell surface, the Notch receptor is proteolytically cleaved by furin-like convertases in the trans-Golgi network, giving rise to two subunits of the mature receptor. The extracellular Notch subunit (ECN) consists largely of a ligand-binding domain composed of small, tandem cysteine knot motifs called epidermal growth factor (EGF)-like repeats (Coffman, 1990, Science 249:1438-1441; Ellisen, 1991, Cell 66:649-661; Weinmaster, 1991, Development 113:199-205; Weinmaster, 1992, Development 116:931-941; Franco del Amo, 1992, Development 115:737-744; Reaume, 1992, Dev. Biol. 154:377-387; Lardelli and Lendahi, 1993, Mech. Dev. 46:123-136; Bierkamp and Campos-Ortega, 1993, Mech. Dev. 43:87-100; Lardelli, 1994, Exp. Cell Res. 204:364-372). In addition to the thirty-six EGF repeats within the extracellular domain of Notch 1, there is a cys-rich domain composed of three Notch Lin Glp (NLG) repeats, which is important for ligand function. This is followed by a cys-poor region between the transmembrane and NLG domain and three LIN 12/Notch repeats that restrain inappropriate, ligand-independent receptor activation.
The transmembrane Notch subunit (NTM) includes a short extracellular domain, a single transmembrane domain, and a large intracellular domain comprised of a an N-terminal domain called the RBP-jkappa associated domain (RAM) and seven iterated cdc10/ankyrin-like repeats positioned between two nuclear localization signals (NLS) (Artavanis-Tsakonas et al., 1995, Science 268:225-232). The ankyrin-like repeat motif is found in many functionally diverse proteins (see, e.g., Bork, 1993, Proteins 17:363-374), including members of the Rel/NF-κB family (Blank, 1992, TIBS 17:135-140), and is thought to be responsible for protein-protein interactions. Lastly, the C-terminal PEST domain is involved in proteosome-mediated Notch degradation and thereby regulates the termination of signaling. In addition, mammalian Notch 1, 2, and 3 contain cytokine response (NCR) regions while Notch 1 and 2 have C-terminal transcriptional activation domains (TAD).
The ligands for the Notch receptors have traditionally been divided into two subclasses, Delta-like and Serrate-like, defined by the absence and presence, respectively, of an additional cysteine-rich domain in the extracellular portion of the polypeptide (Weinmaster, 1998, Curr. Opin. Genet. Dev. 8, 436-442, Zimrin, 1996, J. Biol. Chem. 271, 32499-32502).
Several Notch ligands have been identified in vertebrates, including Delta, Serrate and Jagged. The Notch ligands are also transmembrane proteins, having highly conserved structures. These ligands are known to signal cell fate and pattern formation decisions through the binding to the Lin-12/Notch family of transmembrane receptors (Muskavitch and Hoffmann, 1990, Curr. Top. Dev. Biol. 24:289-328; Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408; Greenwald and Rubin, 1992, Cell 68:271-281; Gurdon, 1992, Cell 68:185-199; Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247; and Weintraub, 1993, Cell 75:1241-1244). A related protein, the Suppressor of hairless (Su(H)), when co-expressed with Notch in Drosophila cells, is sequestered in the cytosol, but is translocated to the nucleus when Notch binds to its ligand Delta (Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247). Studies with constitutively activated Notch proteins missing their extracellular domains have shown that activated Notch suppresses neurogenic and mesodermal differentiation (Coffman, 1993, Cell 73:659-671; Nye, 1994, Development 120:2421-2430).
The mechanism of Notch receptor signaling has been extensively studied and involves the activation of CSL (for CBF1 in mammals, Supressor of Hairless (Su[H]) in Drosophila and Xenopus and Lag-1 in Caenorhabditis elegans)-dependent transcription mediated by the nuclear translocation of the intracellular domain of Notch (Jarriault, 1995, Nature 377, 355-358). Notch displaces corepressors on CSL, changing its function from a repressor into an activator leading to elevated expression of specific genes. Several such targeted genes have been identified and include HES1, a member of the Hairy Enhancer of Split and Hairy Enhancer of Split-Related Proteins families of basic helix-loop-helix transcription factors (Zhou, 2000, Mol. Cell Biol. 20, 2400-2410). However, recent evidence also suggests that Notch signaling may proceed in a CSL-independent manner including pathways utilizing the cytoplasmic protein, Deltex (Small, 2001, J. Biol. Chem. 276, 32022-32030, Nofziger, 1999, Development 126, 1689-1702).
The existence of soluble forms of Notch ligands including the extracellular portion of Delta in Drosophila and other organisms have been reported but their physiological roles have not been determined (Nofziger, 1999, Development 126:1689-1702; Franklin, 1999, Curr. Biol. 9:1448-1457; Hukriede, 1997, Development 124:3427-3437; Han, 2000, Blood 95:1616-1625; Klueg, 1998, Mol. Biol. Cell 9:1709-1723; Morrissette, 2001, Hum. Mol. Genet. 10:405-413; Sun, 1996, Development 122:2465-2474). Transcripts encoding the extracellular domain of the Jagged1 ligand have also been detected in human endothelial cells (Zimrin, 1996, J. Biol. Chem. 271:32499-32502). There is evidence that the Delta ligand can be proteolytically cleaved by Kuzbanian, a member of the ADAM family metalloproteases, to generate a soluble extracellular form (Qi, 1999, Science 283; 91-94), and recent data also suggest that the Notch ligands may be processed by the γ-secretase presenilin in a manner similar to the Notch receptor (Ikeuchi, 2003, J. Biol. Chem. 278:7751-7754).
While studies have suggested that the soluble forms of the Notch ligands are able to activate Notch receptors (Han et al., 2000, Blood 95:1616-1625; Qi et al., 1999, Science 283:91-94; Li, et al., 1998, Immunity 8:43-55), there are numerous reports that the soluble forms of the Notch ligands act as antagonists of Notch signaling by impeding the interaction between Notch receptors and their full-length ligands (Small et al., 2001, J. Biol. Chem. 276:32022-32030; Hukriede et al., 1997, Development 124:3427-3437; Varnum-Finney et al., 1998, Blood 91:4084-4091). Secreted forms of Delta perturb association between full length Delta and Notch (Hicks et al., 2002, J. Neurosci. Res. 68:655-667), and inhibit the Notch-dependent repression of myoblast (Varnum-Finney et al., 2000, J. Cell Sci. 113 Pt 23:4313-4318) and hematopoietic progenitor cell (Han et al., 2000, Blood 95:1616-1625) differentiation in vitro. Likewise, the expression of a non-transmembrane form of the Notch ligand, Jagged1(sj1) also antagonizes Notch signaling in NIH 3T3 cells and induces significant changes in their cellular phenotype including FGFR1-dependent transformation (Small et al., 2001, J. Biol. Chem. 276: 32022-32030; Small et al., 2003, J. Biol. Chem. 278:16405-16413).
Notch signaling plays a key role in normal development through diverse effects on differentiation, survival, and proliferation. These events are highly dependent on signal strength and cellular context (Artavanis-Tsakonas, 1995, Science 268:225-232; Kadesch, T., 2000, Exp. Cell. Res. 260:1-8). Phenotypic analysis of mice null for Notch receptors or their ligands emphasizes the requirement for proper Notch signaling not only during development but in the adult as well (Conlon, 1995, Development 121: 1533-1545; Hamada, 1999, Development 126: 3415-3424; Jiang, 1998, Genes Dev. 12: 1046-1057; Krebs, 2000, Genes Dev. 14: 1343-1352; Xue, 1999 Hum. Mol. Genet. 8: 723-730). Indeed, aberrant Notch signaling has been implicated in several human pathological conditions including the development of the CADASIL (Joutel, 1996, Nature 383: 707-710) and Alagille syndromes (Li, 1997, Nat. Genet. 16: 243-251; Li, 1997, Nat. Genet. 16: 243-251) and the formation of neoplasias in mice and humans (Rae, 2000, Int. J. Cancer 88: 726-732; Rohn, 1996, J. Virol. 70: 8071-8080; Zagouras, 1995, Proc. Natl. Acad. Sci. USA 92: 6414-6418).
The impact of Notch signaling on cell fate and proliferation is determined by cell specific context of its activity. Aberrant Notch signaling can initiate an oncogenic pathway that leads to malignant cell transformation (Maillard, 2003, Immunity 19:781-791; Radtke, 2003, Nat. Rev. Cancer 3:756-767) and tumorigenesis, for example in human T lymphoblastic leukemia (Artavanis-Tsakonas, 1995, Science, 268:225-232), hematopoietic cells (Pear, 1996, J. Exp. Med., 183:2283-2291) and thymic lymphoma (Capobianco, 1997, Mol. Cell Biol., 17:6265-6273). In mammary gland, the transforming effect of Notch4 by virtue of MMTV integration (Jhappan, 1992, Genes Dev., 6:345-355; Smith, 1995, Cell Growth Differ., 6:563-577; Gallahan, 1996, Cancer Res., 56:1775-1785), as well as Notch1ICD and Notch3ICD tumorigenesis in mammary gland have been well documented (Hu, 2006, Am. J. Pathol. 168:973-990).
The earliest events in the pathogenesis of breast cancer typically involve the loss of a normal growth regulatory mechanism by a ductal or lobular epithelial cell. Progression of the disease through the stages of intraductal proliferation to invasive carcinoma, and then to metastatic disease, appears to require additional alterations in growth-regulatory pathways. A substantial body of evidence now supports the idea that these alterations in growth regulation result from genetic events, such as point mutation, deletion, and gene amplification.
One clinical goal is to characterize genetic alterations in breast tumors at the various stages of tumor progression. If metastasis requires additional genetic events beyond those responsible for the intraductal and invasive components of the tumor, one should find genetic alterations in the metastasis that are not present in primary tumor. Alternatively, there may be certain genetic lesions that occur early in tumor development that can predispose a tumor to metastasize without the acquisition of additional genetic defects. The identification of such a lesion would provide an important prognostic indicator, because it would provide a means for predicting the likelihood of the development of metastatic disease in tumors identified at an early stage. The characterization of genetic changes present in individual tumor components thus offers the possibility of identifying new prognostic indicators, as well as helping to elucidate the significance of genetic events to tumor progression.
Notch1, Notch 4 and Jagged 1 have all been found to be increased in human breast cancer tissue (Reedijik, 2005, Cancer Res., 65:8530-8537; Callahan, 2004, 9:145-163). Significantly, high expression levels of Jagged1 and Notch1 correlate with poor patient survival (Reedijik, 2005, 65:8530-8537) whereas Par (2004, Int. J. Mol. Med. 14:776-789) reports that a higher level of Notch 2 expression in breast cancer tissue was correlated with a higher chance of survival. In addition Par demonstrates that Notch 2 was highly expressed in well-differentiated tumors, but poorly expressed in breast tumors with poor differentiation.
What is needed in the art is a clarification of the role of Notch in breast cancer, as well as an overall understanding of the genetic events that transform normal breast tissue to malignancy. In particular, the role of Notch 2 in the development and progression is needed, in order to understand whether Notch 2 can become a target for treatment and intervention of the development of breast cancer. The present invention addresses and meets these needs.